The localization of two mimic epitopes of lipopolysaccharide binding protein and the preparation of their tandem multiple antigen peptide.

Objective: To screen and identify two mimic epitopes in the inflammatory site of lipopolysaccharide binding protein (LBP), and to synthesize and purify their corresponding mimic epitope four branched peptide (multiple antigen peptide, MAP).

Method: Using an anti-full length LBP monoclonal antibody as the target molecule, the amino acid sequences of the exogenous peptides were deduced by combining several different techniques including: affinity screening of the phage display peptide library, the lipopolysaccharide (LPS) binding activity assay and competitive inhibition test, cytokine production inhibition, flow cytometry, and DNA sequencing. Basic Local Alignment Search Tool (BLAST) software was used to compare the resulting peptide sequences with the primary structure sequence of the LBP molecule, and thus the amino acid sequences for two mimic inflammatory epitopes for the binding of LBP and LPS were determined. Additionally, the two target sequences were coupled, and the 9-fluorenylmethyloxycarbonyl (FMOC) solid-phase synthesis method was used to synthesize the 24aa peptide. The design program of the multiple antigen peptide (MAP) was used to couple the four tandem peptides with lysine as the core base to produce the branch like structure, and thus, the four branched peptide was synthesized and purified.

Results: Fourteen phage clones (C) with competitive LPS binding activity with LBP were successfully obtained. Among these, the amino acid sequences of the peptides in C2, C19, C57, C77, C85 and C91 showed a homology of more than 90% to the primary structure of LBP. However, the amino acid sequences of C29 and C90, WKAQKRFMKKSG and LKTRKLFWKYKD, respectively, did not show homology to the primary structure of LBP, which were determined to be mimic epitopes of the inflammatory sites in LBP. Further synthesis of the 24aa peptide using FMOC solid-phase synthesis and MAP modification were carried out, the four branched peptide was synthesized and purified, and the purity was found to be higher than 95%. The purified peptide was subjected to mass spectrometry analysis and amino acid analysis, and its molecular weight (3102.77 kDa) and amino acid composition were in accordance with theoretical values.

Conclusion: The amino acid sequence for two mimic epitopes of the inflammatory site of LBP were determined to be WKAQKRFMKKSG and LKTRKLFWKYKD. The MAP was successfully prepared simultaneously and is able to be used as the core antigen protein for the formulation of vaccines. This knowledge will help in future investigations of the functional characteristics of LBP protein, and enhance exploration into new pathways for the prevention and treatment of LPS inflammatory diseases.