Immunocompetence And Epitopes Mapping Study Of The Major Allergen From Shrimp

With the development of modern society, the structure of people's diet has changed a lot. At the same time, anaphylaxis caused by food shows a rising trend, that is a great threat for people's health. Shrimp products, a kind of crustacean seafood with high value, are deeply popular with consumers because of their delicious flavor and rich nutrition. However, shrimp belongs to one of the eight categories of food allergens and makes a lot of consumers beset by allergy symptoms. This research focuses on the identification of shrimp allergen and the elimination methods of its allergy activity. Further analysis on the epitopes mapping of shrimp allergen was carried out combining with bioinformatic approaches. Finally, amino acid composition of the epitopes was analyzed. The main results are as follows:1. The major allergen protein of Metapenaeus ensis with the molecular weight: 36kD was extracted and separated. Then identification of the major allergen by MALDI-TOF-MS was carried out and its peptide mass fingerprint was obtained. Finally, the figures obtained were identified by searching in the NCBInr database with the Mascot search engine. The results showed that the major allergen of Metapenaeus ensis was tropomyosin. The top one matching protein with the highest mowse value: 268 was the tropomyosin of giant tiger prawn (Penaeus monodon). There were 27 queries matched and the fraction of amino acid sequence coverages was 65%. The matching scores and the sequence coverages were also high with other invertebrates including copramite (Tyrophagus putrescentiae) and silverfish (Lepisma saccharina). The results described above elucidated that the allergen of Metapenaeus ensis had high homology with other crustacean allergens and various invertebrates.2. Three thermal methods were chosen to investigate their impacts on the immunocompetence of shrimp allergen, which included boiling, steaming, and autoclaving. After certain heating time, the total proteins, content of the major allergen and immunocompetence of shrimp allergen were determined. At the meantime, texture profile analysis was adopted to compare the quality changes of shrimp. The results showed that the major allergen of shrimp was still present in the total proteins. However, the immunocompetence of the allergen protein was reduced. There was a new protein in molecular weight of 25-35KD, which took on high immunocompetence. In addition, among these three methods, autoclaving had the best reducing effect towards immunocompetence of shrimp allergen. Cooking 30min with autoclave could reduce 97% of the immunocompetence. Nevertheless, the results of TPA showed that the quality of shrimp had been destroyed during the autoclaving. The parameters of the autoclave were suggested to be optimized to reduce the immunocompetence and keep good texture and nutrition.3. Homology between shrimp allergen and other crustacean allergens was analyzed by bioinformatics approaches. Results showed that the sequences of different crustacean tropomyosins were conservative, which indicated that the high homology of crustacean allergens. Among the selected 14 crustacean allergens, amino acid sequences of Penaeus monodon and Litopenaeus vannanei were exactly the same as that of Pen a 1.Three immunoinformatics tools were used to predict the epitopes and the resultant epitopes were confirmed by Dot-blot inhibition with sera from shrimp allergic subjects. In DNAStar and AntheProt prediction system, five properties of the amino acid sequence were chosen as the parameters for epitope prediction including secondary structure, hydrophilicity, flexibility, accessibility and antigenicity. Furthermore, two prediction websites were also used to predict the epitopes of shrimp allergen. As a result, 10 peptides were predicted and assembled by solid-phase synthesis. 8 epitopes were identified by Dot-blot inhibition test, among which peptide 4 and 8 had not been reported by previous researches. Amino acid analysis demonstrated that Y, E, R, F and S presented more frequently in epitopes. with the three-dimensional structure of pig tropomyosin 1c1gC as the template. And two websites including SWISS-MODEL and CPHmodels were chosen as the homology modeling tools. The modeling conformation was evaluated by Atomic average energy, dynamics simulations and Ramachandran Plot analysis. Results showed that the simulated three-dimensional structure of Pen a 1 had high stability and rationality. It can be seen from modeling result that spacial structure of Pen a 1 was simple, with no complicated three or quaternary structure. The secondary structure was rich in alpha helix. Analyzed from the spacial structure, single chain of Pen a 1 was not easily to from conformation epitopes. Moreover, mapping results of linear epitopes in the special structure showed that linear epitopes were all exposed to molecular surface.5. Key amino acids analysis of Pen a 1 epitopes was carried out by using method of amino acids mutations. The active amino acids of Peptide6 and Peptide10 were substituted by corresponding inactive amino acids of human or pig tropomyosins, using original Peptide6 and Peptide 10 as the refenence. Dot-blot competition enzyme-linked immunosorbent method was adopted to detect the IgE binding activity of these mutation peptides. The results showed that the mutation peptides had lower IgE binding activity when 279S and 278F of Peptide10 was substituted by the corresponding amino acid L of human tropomyosin. As a result, these two amino acids were identified as the key amino acids of Peptide 10. 4. Homology modeling method was used to predict the conformation of Pen a 1...