Allergenicity Assessment Of Hen Egg Iron-free Ovotransferrin And Iron-loaded Ovotransferrin

Food allergy is a global public health problem and egg was considered as the second allergic food.Ovalbumin(OVA),ovomucoid(OVM),ovotransferrin(OVT)and Lysozyme(Lys)were accepted as the main egg allergens in egg white.OVM was considered as one of the most allergic egg allergens because of its resistance to heat and digestibility,while a recently study indicated that the allergenicity of OVT was similar to OVM.OVT is one of the main egg allergens with two iron-binding sites,and iron-loaded ovotransferrin(Holo-OVT)had different tertiary structure compared with iron-free ovotransferrin(Apo-OVT),leading attention to the allergenicity change of OVT.In this work,we assessed the allergenicity of both OVT and Holo-OVT.The main contents including the purification of Apo-OVT and preparation of Holo-OVT;allergenicity evaluation of both Apo-OVT and Holo-OVT through a mouse model;allergenicity evaluation by dendritic cells(DCs)based on antigen process.The main methods,results and conclu,sions are described as follows.1.Apo-OVT was purified by CM Sepharose Fast Flow Cation-exchange chromatography combined with Bio-Gel HT Hydroxyapatite,with a purity of 98%.Then Apo-OVT was dialysised against Fe3+ solution to prepar Holo-OVT.Both Apo-OVT and Holo-OVT were identified by Urea-PAGE,the purity of Holo-OVT was 68%.2.The allergenicity of both Apo-OVT and Holo-OVT were evaluated by a mouse model.Histologic examination of the mouse small intestines revealed that Apo-OVT and Holo-OVT induced intestinal allergy,but no systematic allergeic symbols were observed.Furthermore,compared to Apo-OVT group,the specific IgG and IgGI in seurm,splenic cell proliferation and IFN-?,which was related to food allergy,were decreased significantly.These data indicate that the allergenicity of Apo-OVT was weak,and the allergenicity of Holo-OVT was weaker than Apo-OVT.3.Mouse bone marrow sourced dendritic cells were cultured to analysis the antigen uptake,the results showed that in same time,DCs uptake less Apo-OVT than Holo-OVT.This revealed that antigen uptake by DCs was not positively related to the allergenicity of allergens.4.Cathepsin D was used to simulate antigen process by DCs and both Apo-OVT and Holo-OVT showed no significant difference before and after digestion.These results suggested that compound enzyme should be used to simulate antigen process.Morever,to clearly understand the relationship between antigen process by DCs and food allergy,the type of T cell reponse after antigen primed DCs should be investigated.