Identification And Characterization Of The Novel Allergens From Crayfish(Procambarus Clarkii)

In recent years, the incidence of food allergic diseases grow rapidly. With the consumption of crayfish(Procambarus clarkii) growing and the processed food products diversifying, more cases of hypersensitive reactions caused by crayfish have been reported. It seriously influences the quality of people’s lives. Therefore, it is significant to make the research on allergy to crayfish. This research used crayfish(Procambarus clarkii) as material, based on previous studies, to further study the novel allergens of crayfish, especially finished its purification,physicochemical characterization, and the determination of antigen mimotopes. These will provides the theoretical basis for the development of hypoallergenic crayfish products, and make a great contribution to the diagnosis and treatments of crustaceans allergic diseases.The crustacean-allergic patients’ sera were used for Western-blotting analysis, confirming the 28-k Da protein was the allergen in crayfish. This allergen was purified by ammonium sulfate fractionation and a series of column chromatography(Q-Sepharose and Sephacryl S-200) from crayfish muscle, and identified as triosephosphate isomerase(TIM) by peptide mass fingerprinting. And then anti-TIM polyclonal antibody was made by New Zealand Rabbits. The results of its physicochemical characterization analysis showed that TIM was a glycoprotein with0.82%(w/w) carbohydrate. TIM was unstable in thermal treatment, and high temperature treatment changed its secondary structure detected by circular dichroism spectrum and then reduced its Ig E-binding activity. TIM was resistant to acid/alkali treatment, the alkali treatment could reduce its Ig G-binding activity whereas the acid treatment could increase its Ig E-binding activity partly. TIM was more resistive to simulated intestinal digestion than simulated gastric digestion, but the digested fragments by pepsin remained Ig E-binding activity. TIM had polymorphism, three subunits was detected by two-dimensional electrophoresis named as TIM1,TIM2, and TIM3, meanwhile TIM1 and TIM2 had the Ig E-binding activity obviously. TIM had the typical(β/α)8-barrel structure, and six conformational Ig G mimotopes and five linear Ig G mimotopes were confirmed by phage display technology.Interestingly, a 90-k Da protein had the specific Ig G-binding activity to anti-TIM polyclonal antibody and also had the Ig E-binding activity to crustacean-allergic patients’ sera, so it was predicted as a novel allergen. This protein was purified by ammonium sulfate fractionation andanion exchange column chromatography(Q-Sepharose), and identification as filamin C(FLN c)by peptide mass fingerprinting. The results of its physicochemical characterization analysis showed that the carbohydrates in FLN c were 0.98%(w/w). High temperature treatment changed the secondary structure of FLN c detected by circular dichroism spectrum, and reduced its Ig E-binding activity. FLN c was stable in alkali buffer rather than acidic buffer. It was degraded by simulate digestion, but the digested fragments remained Ig E-binding activity. FLN c had a complicated conformational structure, and eight conformational Ig G mimotopes and nine linear Ig G mimotopes were confirmed by phage display technology. Moreover, there were more than one common mimotopes regions between TIM and FLN c.Based on these results, the interaction between TIM and FLN c was further studied using the phage display technology. It was confirmed that, in TIM structure, there were three conformational binding sites and three linear binding sites to FLN c, and there were eight conformational binding sites and six linear binding sites to TIM in FLN c structure. Therefore,TIM was able to interact with FLN c.