Mapping Of B Cell Epitopes On Major Allergens Bovine ?-lactalbumin

Milk and its dairy products had a high nutritional value, and gain popularity among many consumers. However, milks are one of the eight common food resources causing allergy, which is a common kind of immunological reaction, whose material basis is antigen epitope and antibody paratope. It is reported that ?-lactalbumin is one of the main allergens in milk. But until now, there are limited reports on B cell epitope of ?-lactalbumin. Therefore, the aim of this work was designed to identify the linear and conformational epitopes of bovine ?-lactalbumin.To achieve this, the allergic patient's sera was collected and specific Ig E and Ig G against ?-lactalbumin were purified by affinity purification After that, mimotope were screened from phage random library, Finally, linear and conformtional epitopes were identified by weltool and pymol. The main research method, results and conclusion were as follows:1. 8 seras against bovine ?-lactalbumin were obtained selected from the seras of 19 milk allergic patients by Immuno CAP, and their S-Ig E value were more than 0.35 k UA/l. The specific sera were mixed with equal volume and specific Ig G and Ig E were separated by Sepharose 4B and Protein G immune chromatography column successively and their purity were 93.8% and 85.3%, respectively.2. Mimic peptides were biopanned from phage random peptide library Ph.D.-12 and Ph.D.-C7 C against specific Ig E and Ig G antibody, respectively. 48 clones were randomly selected for linear and comformational Ig G./Ig E mimotopes were obtained by ELISA and DNA sequencing analysis.3. Linear Ig E and Ig G epitopes were identified by Uni Prot software based on aligning with amino acid sequence of ?-lactalbumin. The sequences of 6 linear Ig E binding linear epitopes were AA41-46, AA55-60, AA62-72, AA74-76, AA85-90 and AA92-99, respectively, and the sequences of 7 linear Ig G binding linear epitopes were AA37-46, AA52-54, AA56-59, AA63-72, AA74-76, AA81-90 and AA92-99, respectively. The main amino acids of linear epitopes on ?-lactalbumin protein were aspartic acid and lysine, followed by asparagine, isoleucine and glutamine.4. For conformational epitopes, six Ig E binding and three Ig G epitopes were located thought a Pepitope Server web server which could simulate, calculate and optimize the simulated peptide from phage random peptide library Ph.D.-7C7 and ?-lactalbumin protein amino acid sequence from PDB. The sequences of Ig E binding conformational epitopes were K62-N71-I75- S76-K79,K62-I75-S76-K79-L81,P24-E25-K114-L115-Q117-L119,L105-H107-K108-L110, V42-Q43-S47-T48-Q65-P67 and N44-S47-T48-E49-Y50-K79-L81, respectively. While the sequences of Ig G binding conformational epitopes were F9-R10-K13-K16-L23-P24-L119, I59-Q65-P67-S69-N71-I75 A and Q39-I41-N44-S47-T48, respectively. The main amino acids of conformation epitopes to ?-lactalbumin protein were lysine, leucine, isoleucine and glutamine.