Epitope Mapping, Recombinant Expression And Identification Of Hemocyanin, An Important Allergen In E. Sinensis

Objective:Crab contains a variety of proteins, and each protein can be an allergen. At the same time, in our present study, we confirmed that component sensitization can vary between different patients obviously. Consequently, component-resolved analysis facilitated the production of standardized antigens for in vitro diagnosis and individual immunotherapy. In this study, we found an ignored allergen which played an important role in Eriocheir sinensis allergy. And we analysed the key epitopes of the suspected protein, constructed a recombinant allergen with key epitopes and assessed its immunological property. This study discussed whether the recombinant allergen can be used as a candidate allergen for the diagnosis and treatment of Eriocheir sinensis allergy. Methods:1. Hemocyanin is an important allergen from Eriocheir sinensis: The proteins were extracted from Eriocheir sinensis tissue with lysate extraction method. The protein components were analyzed by SDS-PAGE, 2-D electrophoresis and Western blot. Serum IgE from allergic donors identified a candidate protein, which was characterized by MALDI-TOF/ TOF. The immune property of the candidate protein was tested using dot blot.2. Epitope analysis and key epitopes selection of hemocyanin: We predicted hemocyanin antigen epitopes by three calculation software, and selected ten candidate epitopes which partially overlapped in predictions. Then, we analysed their IgE-binding frequency by dot blot assay. Three epitopes with highest response frequency were selected as key epitopes.3. Recombinant allergen with key epitopes construction and immunological property assession: The gene of the key epitopes were linked and inserted into the pET-28 a vector. Then, the recombinant allergen was expressed in Rosetta competent cell and purified under denaturing conditions with Ni+-NTA affinity column chromatography kit. The full-length recombinant protein Hc was expressed to evaluate the immunity of the recombinant allergen rHc. Finally, we identified rHc by SDS-PAGE, Western blot, and assessed its immunological property by ELISA, dot blot inhibition and ELISA inhibition assay. Results:1. Hemocyanin is an important high-molecular-weight allergen from Eriocheir sinensis: By SDS-PAGE and 2-D electrophoresis analysis, we proved that the native protein components were complete. According to the Western blot result, at least 18 components could react with the positive sera. Among them, two 70 ~ 80 kDa proteins had the same isoelectric point. So, they may be the same protein. MALDI-TOF/ TOF analysis results showed that the suspected proteins were hemocyanin. A sensitization frequency of 61% was observed in Eriocheir sinensis allergic patients by dot blot.2. Epitope analysis and key epitopes selection of hemocyanin: Firstly, ten candidate linear epitopes of hemocyanin were selected according to predicted software. We then showed that ten candidate linear epitopes reacted to at least one of sera from twenty-one patients with allergy to E. sinensis. We selected Epitope 1, 2 and 4, which response frequency over 40%, as the key epitopes.3. Recombinant allergen with key epitopes construction and immunological property assessment: Three key epitopes of hemocyanin were selected to combine into a recombinant allergen rHc according to their response frequency. After expression and purification, the recombinant allergen showed a single major band migrating at about 12 kDa. The recombinant allergen can distinguish allergic and non-allergic serum well(P < 0.01). Dot blot inhibition and ELISA inhibition studies showed that rHc inhibits Hc binding to patients' IgE between 26% and 63%(mean: 50%) and the dose-response curve represents as a sigmoid shape. The binding of the serum to the immobilized Hc was inhibited with 500?g of soluble rHc per millilitre of serum by 93%. Conclusion:1. Hemocyanin is an important allergen with strong allergic property in Eriocheir sinensis.2. Ten candidate epitopes predicted by software are all proved to be antigen epitopes of hemocyanin. Our epitope prediction using the bioinformatics approach is with relative accuracy.3. The recombinant allergen with key epitopes rHc was proved to have well immunological property. And rHc can be a candidate allergen for the diagnosis and treatment of Eriocheir sinensis allergy.