| In recent years,there has been a significant increasing amount of media attention on the subject of food allergy.Among food allergens of animal origin,seafood allergen is a serious cause of adverse reactions in hypersensitive individuals.Nowadays,regular detective methods include ELISA and PCR,which have the disadvantages of high cost,complex operation and low detection accuracy for aquatic products.This study aims to develop an accurate,simple and convenient method for the detection of allergens in aquatic products.At the same time,we use bioinformatics software to predict and comfirm the epitopes of the allergens in Litopenaeus Vannamei and Cyprinus carpio.The main contents are as follows:1.A new method for the determination of tropomyosin in aquatic products by capillary zone electrophoresis was established.The effects of buffer concentration,pH,and separation voltage were carefully investigated.The optimal conditions of electrophoresis are as follows:the detection wavelength was 214 nm,the separation voltage was 15 kV,and running buffer was 25 mmol/L borate buffer(pH=9.2).Under these optimal conditions,the separation and detection of tropomyosin was finished within 5 minutes.A good linear relationship existed for tropomyosin concentration and peak area of electrophoresis in the range from 5μg/mL to 100μg/mL.Linear regression equation was y=16968x+65072 with a correlation coefficient(r)of 0.993.Meanwhile,the LODs(S/N=3)was 3.83μg/mL.The sample added recovery was 91%～103%and the relative standard deviation(RSD)was 8.5%(n=6).Overall,this proposed methodology was rapid,sensitive,and was successfully applied to the quantitative detection of the tropomyosin in aquatic products.2.Capillary electrophoresis(CE)was developed for the quantitative analysis of parvalbumin in aquatic products.The optimal conditions were determined by the multi condition investigation.The optimal conditions consisted of 30 mmol/L borate buffer(pH=9.2),15 kV voltage separation,and 214 nm detection wavelength.The method showed good performance characteristics:a linearity range from 5μg/mL to 100μg/mL(r2=0.994),a detection limits of 2.31μg/mL,and the separation time was less than 5 min.The linear regression equation was y=16464x+38120.The relative standard deviation(RSD)for six injections was 8.5%and the sample added recovery varied from 91%to 116%.This method was successfully applied to the quantitative detection of the parvalbumin in aquatic products.3.Bioinformatics software was used to compare the sequences of 21 kinds of tropomyosin,as a result,tropomyosin is a highly conserved protein that shows high homology in different species.The properties of the amino acid sequences were analyzed to predict the epitopes of Litopenaeus vannamei allergen by DNAStar and AntheProt software.The resultant 10 epitopes were confirmed by IC-ELISA with 10 sera from shrimp allergic subjects.Results showed that 8 epitopes were confirmed by IC-ELISA test,among which peptide 2 had not been reported by previous researches.Moreover,amino acid analysis demonstrated that Glu,Ala,Lys,Leu and Arg presented more frequently in epitopes.4.Using bioinformatics software for sequence analysis of 19 species of parvalbumin showed that parvalbumin is a highly conserved protein and has high homology.Meanwhile,DNAStar and AntheProt software were used to study the two parvalbumin subtypes in Cyprinus carpio.Finally,5 predicted peptides were synthesized and confirmed by IC-ELISA with 10 sera from fish allergic subjects in each subtype.The results showed that 3 epitopes were identified in Cyp c 1.01 and Cyp c 1.02,among which peptide 2 and peptide 6 had not been reported by previous researches.In addition,amino acid analysis demonstrated that Ala,Asp,Gly,Glu and Lys presented more frequently in epitopes of Cyp c 1.01,however,Ala,Asp,Gly and Thr presented more frequently in epitopes of Cyp c 1.02. |
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