Recombinant Expression Of Cashew Allergen Ana O 2 And Establishment Of Cashew Component-resolved Diagnosis

Objective:With the development of allergen component identification and recombinant technology,component resolved diagnosis（CRD）has become an important development direction of food allergy diagnosis.Cashew nut is an important tree nut allergen,which contains three main components,Ana o 1,Ana o 2and Ana o 3.In this study,the prokaryotic expression system was constructed by molecular biology technology.The main allergens of cashew were expressed by E.coli and the immunological activity of the recombinant protein was identified by cashew-allergic sera.Indirect ELISA was established by coating recombinant antigens and the reaction mode and conditions were optimized,the cashew-allergic sera and negative sera were tested,and the molecular diagnosis of cashew allergy was established.Methods:1.The major allergen of cashew Ana o 2 was expressed by prokaryotic system:The amino acid sequence information of Ana o 2 was obtained by querying Gen Bank,and the secondary structure and antigenicity of Ana o 2 were analyzed by bioinformatics software.Ana o 2 sequence without signal peptide was synthesized,and Eco R I（GAATTC）and Xho I（CTCGAG）were added.The optimized Ana o 2 gene and the expression vector p ET-28a（+）were digested with Eco R I and Xho I,and the product was recovered and connected with T₄DNA ligase at 4℃overnight,and the recombinant plasmid p ET-28a（+）-Ana o 2 was transferred into E.coli Rosetta（DE3）sensitive cells.The recombinant Ana o 2 was induced by prokaryotic expression system with IPTG at 37℃for 3 h in large quantities.The recombinant protein was identified by SDS-PAGE and mass spectrometry.The immunological activity of recombinant Ana o 2 was evaluated by Western blot and ELISA.2.To establish the molecular diagnosis method of cashew allergy:Indirect ELISA was established by coating three main allergentic components and optimizing the reaction mode and conditions.The goat anti human Ig E antibody labeled with horseradish peroxidase was used as the second antibody to establish the molecular diagnosis method of cashew allergy.The cashew-allergic sera with clinical symptoms and history of cashew allergy,serum total Ig E>100 k U/L,cashew s Ig E>0.35 k U_A/L and negative sera were collected and tested respectively.The binding rates of cashew allergen components and the sera s Ig E response heterogeneity of cashew-allergic patients were analyzed.According to the s Ig E level of cashew allergen components,evaluation of the effect of the components on the diagnosis of cashew allergy by binary logistic regression,and the Receiver Operating Characteristic curve（ROC curve）was established to evaluate the diagnostic value of cashew components protein.In order to further optimize the diagnosis efficiency,the application of multi-components protein combined diagnosis in cashew allergy was further evaluated by the ROC curve of combined variables.Results:1.The optimized Ana o 2 sequence without 1-14 aa signal peptide region was constructed into expression vector p ET-28a（+）.The recombinant plasmid was transferred into E.coli Rosetta（DE3）.After 3 h of induction by 1 m M IPTG,the target protein band was identified by SDS-PAGE at the theoretical value of 54 KD.The molecular weight of the recombinant protein was 51.4 KD and the isoelectric point was 6.27 by mass spectrometry.The result of comparison with the target protein Ana o 2（Gen Bank:aan76862.1）was acceptable.Western blot showed that the recombinant Ana o 2 had obvious specific binding with the positive cashew allergy sera.The results of ELISA showed that there was a significant difference in the level of recombinant Ana o 2-s Ig E between cashew-allergic sera and negative sera（P<0.05）.Recombinant Ana o 2 could distinguish the positive and negative sera,and could be used as a known antigen for the preparation of single-component-s Ig E antibody detection reagent.2.Single component of cashew allergen was directly coated on polystyrene 96well enzyme plate at the final concentration of 1μg/m L,then the sera with 1:20dilution and the goat anti-human Ig E antibody labeled with horseradish peroxidase（HRP）with 1:2 000 dilution were added in turn,and finally the HRP substrate was added.The binding rates of Ana o 1,Ana o 2,Ana o 3 to sera s Ig E of cashew hypersensitivity were 29.63%,51.85%and 57.41%.There was a significant difference in s Ig E levels between the cashew allergy positive and negative sera（χ²=9.385,P<0.05）.There was no significant difference between the sexes（χ²=0.394,P=0.831）.According to the ROC curve,the Area Under Curve（AUC）of Ana o 3 was0.922,which is the most important index in the single component diagnosis of cashew allergy.The AUC of multi-components combined diagnosis was higher than that of single component detection.When the three components combined diagnosis was used at the same time,the AUC could reach 0.969,which was the most effective combination for the detection of cashew-allergic sera.Conclusion:1.The recombinant Ana o 2 was successfully expressed by prokaryotic system.It has high yield and purity.It has good reactivity with cashew-allergic sera,and can distinguish the positive and negative cashew-allergic sera,which provides useful materials for the component diagnosis of cashew allergy.2.The component resolved diagnosis method of cashew hypersensitivity was established by indirect ELISA using cashew single component protein.The binding rates of cashew components and s Ig E heterogeneity in patients with cashew hypersensitivity were analyzed.The value of each component protein in the diagnosis of cashew allergy was evaluated by ROC curve.The multi-components combined diagnosis can further improve the detection efficiency.