Analysis of the Allergenic Epitopes of Tropomyosin from Mud Crab Using Phage Display and Site-Directed Mutagenesis

Abstract

Mud crab ( Scylla serrata), which is widely consumed, can cause severe allergic symptoms. Eight linear epitopes and seven conformational epitopes of tropomyosin (TM) from S. serrata were identified using phage display. The conformational epitopes were formed based on the coiled-coil structure of TM. Most of the epitopes were located in the regions where primary structures were conserved among crustacean TM. Twelve synthetic peptides were designed according to the epitopes and trypsin-cutting sites of TM, among them, three synthetic peptides (including one linear epitope and two conformational epitopes) were recognized by all of the patient sera using inhibitory dot blotting. A triple-variant (R90A-E164A-Y267A) was constructed based on the critical amino acids of the TM epitope. The IgE-binding activity of the triple-variant was significantly reduced compared with that of native TM. The results of phage display and site-directed mutagenesis offered new information regarding conformational epitopes of TM.

Production and characterization of monoclonal antibodies against Toxoplasma gondii ROP18 with strain-specific reactivity

“…Thus, the identification of an antigen epitope is of great significance in studying the whole process of immune response and understanding the structure and function of the antigen itself. The traditional methods of epitope identification include peptide scanning (Chen and Chang, 2017), site-directed amino acid mutagenesis (Liu et al ., 2018), phage display (Liu et al ., 2018), immunoaffinity mass spectrometry (Purcell and Gorman, 2004) and bioinformatic prediction technologies (Mei et al ., 2019). Among these methods, peptide scanning has the advantage to be convenient and accessible, but it can only identify linear epitopes.…”

Progress in the Analysis of Food Allergens through Molecular Biology Approaches

“…In a first study, 46 overlapping peptides, 15 amino acids long and covering the whole tropomyosin of shrimp ( Penaeus aztecus ), were analysed by immunoreaction with the sera from 18 shrimp allergic subjects, allowing the identification of five major linear epitopes [41]. In a more recent article by Liu et al [42] both the approaches (synthetic peptide and phage display screening) were used to identify epitopes in the tropomyosin molecule of mud crab ( Scylla serrata ). A group of 14 mimotopes (putative epitopes identified by phage display) were firstly identified by screening a commercial phage display library against a rabbit anti- S. serrata polyclonal antibody.…”

Effects of Food Processing on the Stability and Quality of Shellfish Allergens

“…Past studies highlighted the modification of the food allergenicity based on the heat treatment process and Maillard reaction. The result of these reactions had proven that thermal processing has an influential impact on the proteins' allergenicity whether it can be reduced by reducing the epitopes or enhanced by exposing the epitopes [25].…”