| Hen's egg allergy is the common food allergy in infants and young children. It affects 0.5% to 2.5% of the young children. Ovotransferrin (OVT) is one of the five major allergenic proteins identified in the egg of the domestic chicken (Gallus domesticus), and has the second amount in egg white. It can be recognized by 22% of egg-allergic patients. Currently, the egg allergic mechanism caused by OVT is still unclear, one of the reason is that we have no idea about the epitopes on OVT. As the commercial OVT contains some impurities with a high price, how to purify OVT simply, fast and economically plays an important role in the investigation of OVT allergic mechanism. In this work, a simple protocol for OVT purification with high purity was developed and the allergenicity and antigenicity of prepared OVT were evaluated by using rabbit polyclonal antibody against OVT and egg-allergic patients' sera. Subsequently, heptapeptide phage display peptide library was used for panning the minotopes of OVT by using purified rabbit polyclonal antibody as the target molecules. DNAStar software, bioinformatics resource portal ExPASy and online database ADFS (allergen database of food safety) were chose to predict the linear epitopes and IgE binding epitopes on OVT. The potential linear epitopes on OVT was identified by the comprehensive analysis of the predicted epitopes and minotopes. Finally, the prepared OVT was heated by different temperatures and times to assess heat induced changes of the potential allergenicity and the structure of OVT. It gave an insight into mechanisms of possible changes of allergenicity of OVT by heating, and also provided theoretical and technical basis for developing the hypoallergenic or non-allergic egg products. The main results and conclusions presented as follows.1. One single step DEAE-Sepharose Fast Flow anion exchange chromatography was developed for purifying OVT from egg white. It was shown that this innovative procedure was efficient for egg OVT purification with high purity (97%), high recovery rate (87.47%), relatively low cost and easy performance, and it provided a good material for the following work. 2. The physicochemical and immunological properties of prepared OVT were well defined. The molecular weight of prepared OVT was 77,726 Da, the pI value was 6.85, and its structure was proved to be folded. The specific IgG and IgE binding ability of prepared OVT was very sensitive and strong.3. Specific rabbit polyclonal antibodies were obtained from New Zealand rabbits by injection of prepared OVT, with the titers of higher than 1:800,000. The analysis of indirect ELISA, competitive inhibition ELISA and western blotting showed that the antigenicity and allergenicity of prepared OVT were maintained well throughout the purification procedure, which further validated the reliability of the one-step purification procedure for OVT.4. An affinity chromatograph approach was developed to purify the specific rabbit anti-OVT IgG with high purity. The prepared OVT was coupled to CNBr-activated Sepharose 4B with the amount of 5mg protein per millilitre Sepharose 4B. The specific rabbit antibody was eluted with 3 mol/L MgCl2. The SDS-PAGE profile showed that antibody has high purity, and the ELISA analysis of its titer was 1:200,000.5. A total of 274 positive clones were identified and carried out DNA sequencing among 480 random clones derived from the panning of heptapeptide phage display peptide library with purified anti-OVT rabbit sera. The alignment analysis of the deduced amino acid of the positive clones was done using the web tool of Clustalx, followed by identification 17 mimic epitope regions on OVT, including AA1-3, AA138-140, AA214-219, AA292-295, AA306-312, AA318-320, AA322-325, AA392-394, AA415-418, AA444-445, AA464-468, AA479-484, AA531-533, AA568-570, AA592-596, AA625-628 and AA660-662.6. To define the linear epitopes on OVT, bioinformatics analysis was performed by using DNAStar software and web service ExPASy. The antigenic index, hydrophilicity, surface probability, flexibility and the secondary structure of OVT were predicted, respectively. Based on the comprehensive analysis of the predictions, 17 possible regions on OVT, including AA13-16, AA85-90, AA 174-181, AA215-222, AA231-240, AA256-258, AA277-280, AA288-294, AA332-344, AA415-429, AA438-442, AA491-495, AA517-519, AA547-555, AA598-600, AA619-622 and AA648-652 were predicted as the main linear epitopes.7. The information on IgE binding epitopes on the prepared OVT was predicted using online database ADFS (allergen database for food safety) by blasting the amino acid sequence with IgE binding epitopes sequence of 98 epitope-known allergens, followed by identification of 45 possible IgE binding epitopes on OVT.8. Based on the alignment among the predicted epitopes obtained from the software and ADFS, and the minotopes from the heptapeptide phage display peptide library,14 of potential human IgE binding linear epitopes on OVT,3 of potential rabbit IgG binding linear epitopes on OVT, as well as 10 of potential linear epitopes shared by IgG and IgE binding were preliminarily predicted in this study.9. Heat induced changes of the allergenicity and the structure of OVT were clarified. Changes in conformational structure of OVT were characterized by circular dichroism (CD) spectroscopy, ultraviolet (UV) absorption spectroscopy and fluorescence spectroscopy. And the changes in the allergenicity of OVT were measured by ELISA. The allergenicity of OVT increased with the folding of the structure which exposed hydrophobic regions onto the surface of the molecule, while decreased with the cleavage and the rearrangements of disulphide bonds.10. The severity of heat treatment affected the changes of allergenicity and the structure of OVT. The effect of reducing the allergenicity was dependent on the degree of treatment and the characteristic of patients that whether they can tolerate cooked egg or not. For some egg allergy patients, it is maybe a good way to reduce the allergenicity of heated ovotransferrin by controlling the heating temperature and time.
|
PDF Full Text Request