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Epitope Mapping of Bovine a-lactalbumin

Author: WenXueFang
Tutor: ChenHongBing
School: Nanchang University
Course: Biochemical Engineering
Keywords: cow milk allergy a-lactalbumin phage display epitope
CLC: TS252.1
Type: Master's thesis
Year: 2010
Downloads: 46
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Abstract


Milk and milk products are nutritious food items containing numerous essential nutrients which are consumed by many people, but, at the same time, belong to one of the common food allergens. Milk allergy affecting 2-2.5% of children and 1% of adult population and have a severe impact on the healthy and quality of life who suffer from it. Therefore, the investigation on milk allergy may help to better understanding and preventing it.Bovine a-lactalbumin belongs to the lysozyme superfamily which was also considered as one of the major allergens in milk. The current work was composed of four parts including purification of bovine a-lactalbumm,generation and purification of anti-serum against bovine a-lactalbumin, mapping of linear and conformational epitopes on bovine a-lactalbumin.1. Purification of bovine a-lactalbumin utilizing DEAE -Sepharose Fast Flow anion exchange chromatography and Sephadex G-75 gel chromatography followed by SOS-PAGE. The SDS-PAGE identification showed that the bovine a-lactalbumin obtained has a purity of up to 90%, with a recovery rate of 19.42%2. Two Japanese rabbits were immunized with purified bovine a-lactalbumin following the routine approach to develop anti-sera. The titers of the specific serum gained were 1:204,800 and 1:320,000 corresponding to rabbit A and B,respectively by indirect ELISA, and no cross-reactivity was found between a-lactalbumin and other milk proteins, which indicating a good specificity of the sera. However, western blotting showed varied degrees of cross-reactivity between bovine a-lactalbumin and a-lactalbumin from other species.3. For the purification of specific antibody against bovine a-lactalbumin, affinity chromatography was carried out with bovine a-lactalbumin as a ligand coupled to CNBr activated Sepharose 4B. Following being eluted with 3M MgCl2, highly purified antibody was gained. The indirect ELISA revealed that the titer of purified sera was well preserved during purification.4. The Ph.D-7 and Ph.DC7C phage display peptide library were used to select specific phage clones binding to purified IgG. After 4 rounds of panning, random selected phages were checked by ELISA, and the amino acid sequence of inserted peptides were deduced by sequencing DNA of positive clones. The alignment of the sequences selected by Ph.D-7 revealed the linear epitope regions binding to rabbit IgG on bovine a-lactalbumin was located in the AA21-26, AA36-41, AA63-68, AA79-82, AA114-119. While by utilizing bioinformatics methods, mimotopes obtained from Ph.D C7C were matched to two regions on bovine a-lactalbumin which corresponding to T33 S34 Q39 A40 Q54 and D46 S47 N56 D63 D64 Q65 N66 P67 H68 S69 S70 N71,respectively.

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