Dissertation
Dissertation > Medicine, health > Internal Medicine > Systemic disease > Autoimmune diseases > Allergic diseases

Characterization of the Allergens from Crayfish (Procambarus Darkii)

Author ChenHengLi
Tutor LiuGuangMing
School Jimei University
Course Microbiology
Keywords Procambarus clarkii Allergens Identificaiton Purification Physicochemicalproperties Molecular properties
CLC R593.1
Type Master's thesis
Year 2013
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Recently, with the increasing production and consumption of crayfish Procambarus clarkii,more cases of hypersensitive reactions after its ingestion have been reported. It influences theeating safety of crayfish and the health of people. Therefore, it is very urgent and meaningful tomake the research on allergy to crayfish. This study used crayfish (Procambarus clarkii) asmaterial, analyzed the allergens of crayfish, especially finished the purification and propertiescharacterization and investigated the changes of allergens under different processing treatments.These will make a contribution to developing the hypoallergenic or non-allergenic products.The detection of the allergens of crayfish was conducted by Western-blot using27cases ofcrustacean-allergic patients’ sera. The result showed that the three proteins with the molecularmass of36kDa,40kDa and22kDa were the allergens in crayfish.The40kDa allergen was purified by ammonium sulfate fractionation and columnchromatography from crayfish myogen. It was identified as arginine kinase (AK) byimmunoblotting using rabbit anti crab AK polyclonal antibody. The results of properties analysisshowed that AK was a glycoprotein with0.60%carbohydrate. AK was unstable in thermaltreatnment, and its IgE-binding activity could be reduced by high tempertature treatment. AKwas easily degraded in acidic buffer and alkali treatment could also reduce its IgE-bindingactivity. AK was more resistive to pancreatic juice digestion. AK had a complicatedconformational structure and linear epitopes and conformational epitopes were both existed inAK. Boiling treatments could be effective to reduce the IgE-binding activity of AK.The22kDa allergen was purified by ammonium sulfate fractionation and columnchromatography from crayfish myogen. It was identified as sarcoplasmic calcium-bindingprotein (SCP) by mass spectrometry. The results of properties analysis showed that SCP was aglycoprotein with4.90%carbohydrate. SCP was stable in the processes of thermal andacid-alkali treatment, and its IgE-binding activity was not decreasing, but it could be digested bysimulate gastrointestinal fluid. SCP had polymorphism and SCP-II had the lower IgEbind-activity. Three kinds of subunits a, b and c of SCP were all had IgE-bind acitivity. SCP waseasy to form epitopes since it had a lot of Turns and Coils.The36kDa allergen was purified by preparation of acetone powder, isoelectricprecipitation, ammonium sulfate fractionation and column chromatography.from crayfishmyofibril. It was identified as tropomyosin (TM) by immunoblotting using rabbit anti crab TMpolyclonal antibody. The results of properties analysis showed that the carbohydrates in TM were1.14%(w/w). TM was stable in the processes of thermal and acid-alkali treatment, but itwas less stable simulated digestion. TM was a superhelix consisted of α-helices and was full oflinear epitopes.Myosin light chain (MLC) and hemocyanin (HMC) were the novel allergens of shrimp. Inthis study, the two proteins were firstly purifed and were further confirmed by nire cases ofcrustacean-allergic patients’ sera.The MLC was purified by preparation of acetone powder, isoelectric precipitation,ammonium sulfate fractionation and column chromatography from crayfish myofibril. Itrevealed a single band with molecular mass of18kDa and further confirmed by massspectrometry. Analysis using sera from crustacean-allergic patients showed that MLC wasprobably an allergen of crayfish. Analysis of the properties showed that the carbohydrates inMLC were4.30%(w/w). It was stable in the processes of thermal and acid-alkali treatment, butit could be digested by simulate gastrointestinal fluid. MLC was consisted of α-helices and Coils.Linear epitopes and conformational epitopes were both existed in MLC.The HMC was purified from crayfish hemolymph via centrifugation and chromatographyand it revealed six submits with molecular masses of68,72,76,82,84and88kDa onSDS-PAGE. HMC was further confirmed by rabbit anti shrimp HMC polyclonal antibody.Analysis using sera from crustacean-allergic patients showed that HMC was also probably anallergen of crayfish. The results of the properties showed that he carbohydrates in HMC was1.99%(w/w). It was stable to thermal treatment and SIF digestion, but less stable to acidic treatment.In conlusion, TM, MLC and SCP of crayfish had the similar physicochemical properties,they were all stable to thermal and acid-alkali treatment, and could be digested by simulategastrointestinal fluid. AK and HMC shared the common properties. They were both unstable toacid but resistive to SIF digestion. The conformational structures of AK, SCP and MLC weremore complicated, than TM. The sera identification and properties characterization showed thatTM was the main allergen of crayfish, AK and SCP were the important allergens of crayfish,while MLC and HMC were the protential allergens of crayfish. However, there may be otherallergens in crayfish too.

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