Identification And Analysis Of Major Alergens In Chinese Shrimp, Fenneropenaeus Chinensis

The aims of this work were to establish an allergic model in mice by shrimp protein sensitization and identify the major allergens in Chinese shrimp, Fenneropenaeus chinensis. The protein was extracted by liquid nitrogen grinding in a lysis buffer. Intraperitoneally injection of protein extracts was used to stimulate allergic responses in ICR mice. Moreover, shrimp major allergens were identified by two-dimensional immunoblotting using sera from subjects with shrimp allergy, followed by matrix-assisted laser desorption ionization time-of-flight mass spectrometry analysis of the peptide digest. Finally, a specific allergen, Chinese shrimp tropomyosin was enriched from protein extracts and analyzed in P815 mast cell sensitization model. The results are listed as follows:1. Considering the potential harmfulness of protein extraction buffer in vivo, we performed two different extract methods and submitted to two-dimensional electrophoresis for optimization as well as injection of mice for allergic model establishment. Intraperitoneally injection of protein extracts was used to stimulate allergic responses in ICR mice. The indirect ELISA was employed to test the levels changes of specific IgE and IgGl antibodies in sera of mice, and the symptom scores were evaluated during different stages of the challenging period. The allergic symptoms showed in a dose-dependent manner in mice, and injection of high dose (1.0 mg) of protein can significantly (P<0.05) increase the levels of specific IgE and IgGl in sera and contribute to the obvious symptoms compared with the control. Protein extracts from both methods can evoke allergic reactions in ICR mice, indicating that sensitization model has been established.2. The optimized conditions for separating Chinese shrimp proteins in two-dimensional electrophoresis system are as follows:80000vhr, 12.5% separating gel, and passive hydration. Then, the potential allergens were identified by the two-dimensional immunoblotting. Two kinds of allergens were identified as arginine kinase (MW:40.2 kDa, PI:6.0 and 6.2) and tropomyosin (MW:35.8 kDa, PI:4.6) in the species of Chinese shrimp by MALDI-TOF/TOF-MS and Mascot database research. By alignment, these two proteins have up to 95% homology with crustaceans arginine kinase and tropomyosin, which indicates the major allergens in Chinese shrimp. We also speculated that Chinese shrimp arginine kinase may have protein modification post translation which leads to the presence of two spots with different pI points.3. The Chinese shrimp tropomyosin was isolated and purified by salting out. By SDS-PAGE analysis, the enriched tropomyosin showed 95% purity with molecualar weight of 36 kDa. The purified tropomyosin had 90% similarity compared to the previously identified tropomyosin by mass spectrometry. Finally, the effects of temperature, pH and in vitro mimic gastrointestinal conditions on the stability and sensitization of tropomyosin were investigated in the P815 mast cell model. It was found that tropomyosin is a heat- and acid-resistant protein, which can be partly degraded by pepsin, trypsin and chymotrypsin and reduced its allergenicity.