| Egg is recognized by FAO as one of the eight main allergic foods, besides, it is one of the most important sources of food proteins for human. Egg proteins are the best protein with its biological value up to95. Egg Ovotransferrin, which is also called OVT, contains about12%of egg white protein, and it is well known as a major allergen in egg white. OVT is also an iron-binding monomeric glyprotein with a function of binding and transporting iron ions, and it is easy to chelate with the iron ions in solution. Therefore, this work aimed to define the structural and allergenicity changes in the process of OVT chelating iron ions, and the digestibility of ovotransferrin was investigated, contributing to clarify the relationship between structure and allergenicity of egg OVT, and it will provide information about the effect of metal ions on the structure and allergenicity of OVT.Egg allergen OVT was separated and purified, followed by preparing the Holo-OVT and Apo-OVT, and these two proteins were used as antigens to immunize rabbits to obtain the corresponding antibodies. In the process of combined and got off iron ions, its conformational and potentially allergenic changes were evaluated. Moreover, the digestibility of Apo-OVT and Holo-OVT were evaluated by simulated gastric fluid (SGF) and simulated intestinal fluid (SIF). The major findings and conclusions are described as follows.Firstly, the egg allergen OVT were purified by anion exchange chromatography combined with and hydrophobic interaction chromatography with the purify up to98%by SDS-PAGE, followed by preparing Apo-OVT and Holo-OVT using methods such as dialysis and ultrafiltration, and the Apo-OVT are iron(Ⅲ) free, while Holo-OVT contain about90percent of two-iron(Ⅲ)-chelated OVT molecules.Secondly, Apo-OVT and the Holo-OVT were used as antigens to immunize New Zealand rabbits separately, and the corresponding polyclonal antibodies were prepared, followed by checking the titer and specificity by indirect ELISA and Western-blotting separately. The titer of anti-Apo-OVT polyclonal antibody and that of anti-Holo-OVT polyclonal antibody are up to1:200,000and1:100,000, respectively, and the specificity is highly enough to meet the need of evaluating IgG-binding ability.Thirdly, the process of binding iron was simulated by adding iron ions to the Apo-OVT solution slowly, and the process of iron removal from Holo-OVT was simulated by decreasing the pH of the protein solution. Then, far-UV circular dichroism, UV spectroscopy and intrinsic fluorescence spectroscopy were used to define the protein conformational changes, and indirect and competitive inhibited ELISA were used to detect the potentially allergenicity changes. It was shown that the secondary structure of OVT did not change significantly during the binding or removing of iron(Ⅲ). Apo-OVT chelated OVT slowly when the iron(Ⅲ) concentration increased, and the tertiary structure was unfolded at first, followed by folding tightly with a reduced IgE binding capacity. In respect to the pH value, the Holo-OVT started to release iron ions when the pH value of the Holo-OVT solution was decreased, and the protein’s tertiary structure unfolded with reduced IgG and IgE binding capacity.Finally, the digestibility of Apo-OVT and Holo-OVT was assessed with SGF and SIF, and the assessments were defined by SDS-PAGE analysis and IgG binding ability evaluated by competitive inhibition ELISA. It was shown that the digestibility of Apo-OVT is weak, and Holo-OVT is even more easily digested by simulated gastric and intestinal fluid. Moreover, the digestibility of both Apo-OVT and decreased when increasing the pH value of SGF. As for IgG-binding ability of Apo-OVT and Holo-OVT, both of them became lower when increasing the digestion time of SGF and SIF. |
PDF Full Text Request