Conformational Epitopes Mapping Of Bovine β-casein

Bovine milk is one of the eight common allergenic foods, it is essential in the different territories and different dietary habits. It is reported that the milk allergy is more prevalent during children, and the incidence in the children aged below 1 year is about 2-3%. Undoubtedly, the research on the milk allergy is important to the improvement of the living quality of the people who suffer from milk allergy. Since epitope is the binding sites of antibody and antigen, allergen epitopes will play an important role in food allergens, and epitope mapping should be one of the key problems in the milk allergy research.This work focused on beta-casein, one of the magor allergic proteins in bovine milk, and it includes the purification and separation of the beta-casein, the preparing of specific rabit serum against beta-casein, the purification of the polyclonal antibody by affinity chromatography, modelling and epitopes mapping of the beta-casein. The detailed process and conclusions are presented as follow.1. Crude caseins were prepared from fresh milk using the PI precipitate method, followed by lyophilization, and the dried caseins powder was then loaded into the columns packed with the DEAE-Sepharose Fast Flow anion exchange medium. The target eluent then identified by the SDS-PAGE and IEF PAGE, and it shows the purity of isolated beta-casein was higher than 90%.2. Based on the routine way of immunization, specific rbbbit serum against beta-casein was produced. An indirect ELISA approach was developed to check the change of immune response titer of two rabbits, and the final antiserums titer are 1:240,000 and 1:150,000, respectively.3. The antiserums loaded to Sepharose4B which actived by CNBr, the purified components then identified by SDS-PAGE, the result illustrated two bands with the molecular weight were 55 Kda and 25 Kda respectively; Also the indirect ELISA be carried through to the purified antibody, then it is come to the conclusion that the polyconal antibody with a higher specificity and higher purity;4. The beta-casein models were obtained using the homology modeling method, fold recognition and ab initio medhod, the models then were optimized and assessed by Ramanchandran plot, WHAT_CHECK, ERRAT and the like, the most optimal model compared with the known model created by Kumosinski et al, in this process, the experimental data were used as reference and benchmark, from the results, the conclusion can be drawed that homology model is the most optimal model, and this model is more identical with the experimental data than Kumosinski et al's model;5.6 mimic peptides obtained after the biopanning process, on the other hand, the antibody-antigen complexes which the 3D structures were resolved were filtered, then the epitopes information from the complexes were extracted to constitute the epiotpes information database, the information such as the number of the amino acids included in the epitopes, the amino acids preference in the amino acids, the amino acids which play a interval role in the epitopes, the area the epitopes extend and the like are analysed, finally all these information combined to the pepitides that got from the phage display method for the analysis of epitopes, then four conformational epitopes were obtained in this research.