The Identification And Property Studies Of Antarctic Krill (Euphausia Superba) Allergens

Antarctic krill(Euphausia superba)is a type of crustacean living in Antarctic waters and is an import ant strategic marine living resource.Currently,the research on the safety of Antarctic krill is mainly focused on fluorine and arsenic,while the study on Antarctic krill sensitization is rarely reported.In this thesis,krill protein was extracted from shelled krill meat with salt solution and analyzed by SDS-PAGE;Western blot(WB)was used to screen the allergens of Antarctic krill by cross reaction between serum of patients with shrimp and crab allergy and krill protein;The primary structure of these allergic proteins were identified using high performance liquid chromatography-tandem mass spectrometry(HPLC-MS/MS);Major allergens were separated and purified by isoelectric precipitation,ammonium sulfate salting out and anion exchange column chromatography;The heat resistance and the digestive stability of Antarctic krill major allergens to simulated gastrointestinal fluid were analyzed by SDS-PAGE and WB;The mouse model of promyosin sensitization was established,and the sensitization of the sensitized protein was evaluated by the indexes of allergic behavior,immunoglobulin E(Ig E),specific Ig E of the sensitized protein,histamine and splenic cytokines.The main results are as follows:(1)Sarcoplasmic protein(MS)and myofibrin(MF)were extracted from krill meat by salt extraction method.Sds-page analysis showed that the bands of each protein were abundant and the molecular weight range was wide.Antarctic krill proteins could react with the serum Ig E of shrimps/crabs allergic patients,and at least four proteins had positive reactions.The protein with the strongest immune reaction has a MW of about 35 k Da and can be recognized by the serum of all allergic patients.(2)The protein was preliminarily purified by isoelectric point precipitation,ammonium sulfate salting out,and purified by anion chromatography column.The optimal saturation of TM purification by fractional salting out with ammonium sulfate is 50%.After purification by anionic chromatography,TM with high purity was obtained.It has good thermal stability and can have strong immune reaction with serum of shrimp and crab allergy patients.(3)The TM isoelectric point of krill was 4.4;SDS-PAGE analysis was performed on TM stored at 4℃ after heated at 40℃,60℃,80℃ and 100℃ water bath for 30 min,The results showed that TM did not decompose,indicating that TM has strong heat resistance.Porcine pepsin configuration was used to simulate gastric juice,The optimum saturation of ammonium sulfate was 50% for the salting out Antarctic krill TM.After separation and purification,TM with high purity was obtained,which could have a strong immunoreactivity with the serum Ig E of shrimps/crabs allergic patients.The measured isoelectric point of Antarctic krill TM was 4.4,and it had good thermal stability.Porcine trypsin configuration was used to simulate intestinal fluid,With the extension of digestion time of simulated intestinal fluid(SIF),the original band of TM gradually decreased until disappeared,and finally completely degraded into smaller peptides.The immunoreactivity of TM degradation products was greatly reduced after digestion by SIF.(4)The hydrophilicity,plasticity and antigenicity of Krill TM were predicted by bioinformatics method.The predicted results showed that TM had high hydrophilicity and plasticity,and the amino acids with β-rotation were exposed on the surface of antibody and more likely to bind to antigen,and the amino acid sequence was 101-107;120-126;140;148-150;159-165;178;180-189;212-224;231;234-236;248-257;266-270;273;275-278 may be the potential epitope of TM.The mice were sensitized with promyosin by intraperitoneal injection.The hypersensitivity of TM in mice proved that TM had strong sensitization.There are 19 figures,10 tables and 100 references in this dissertation.