Identification And Structural Characterization Of Conformational Epitopes On Bovine β-lactoglobumin

Milk allergy is one of the most serious food allergy problem, and general population birth cohorts reported a cow milk allergy prevalence of2.2%to2.8%at1year of age. There are more than20proteins in milk. However,β-lactoglobulin is one of the main allergens, and about82%of milk allergic patients are sensitivity to bovin β-lactoglobulin. Moreover, epitopes involve linear and conformational epitopes, and they are the biding sites with antibody. Comparing to linear epitopes, conformational epitopes is not well known, resulting in the necessity of identification of conformational epitopes on bovin β-lactoglobulin.This research focused on bovin β-lactoglobulin, and its practical work include the purification of anti-β-lactoglobulin IgE, the monitope selection by phage display technology, modeling and epitopes mapping of the bovin β-lactoglobulin, and the analysis of structural characteristics. The main results and conclusions are presented as followes.1. The specific IgE from the patients' serum was purified by affinity chromatography on Sepharose4B and Protein G. The purity and specificity of the IgE against bovin β-lactoglobulin was defined by ELISA.2. Three conformational monitopes were produced through3round specific selection by phage display, and the mimic peptides are DYSEHKE, TIPGQGP, NQKLSNL, respectively.3. The analysis of the mimic peptides was carried out using the web tool of Pepitope, and three conformational epitope on bovin β-lactoglobulin were produced, which are Leu87-Asn88-Asn90-Lys91-Glu108-Ser110, Ile2-Thr4-Pro113-Gln115-Pro144,Thr18-Tyr20-Glu44-Glu45-Lys47-Glu55-Glu157, respectively.4. With the help of bioinformation software DNAStar and SOPMA service, the structural characteristics of the three identified conformational epitopes on bovin β-lactoglobulin were analysed, and it was shown that the most of the amino acid on the epitopes are polar amino acids. Based on the characterization of the primary structure defined by DNAStar, three, two and seven amino acids of the conformational epitopes were on the hydrophillcity plot, the surface probability and antigenic regions, respectively. While on the basis of the predicted secondary structure, seven, six and five amino acids of the conformational epitopes were on the a-hilex, the β-sheet and the coil, respectively. However, two, eight and eight amino acids of the conformational epitopes were on the P-sheet, the β-turn and the coil, respectively when the three epitopes were modled on the3-D structure of the bovine β-lactoglobulin.