Epitope Mapping,Recombinant Expression And Identification Of Cashew Nut Allergen Ana O 3

Objective:Allergen-specific IgE testing in human serum is one of the most important methods for food allergy diagnosis.Currently,it has advanced into the stage of molecular diagnosis.Epitope is the basic unit of antigen that is specifically recognized by antibody.Through analyzing the epitope profile of an allergen,accurate detection of s Ig E in serum can be realized.Therefore,in this study,Ana o 3,taken as the research object,was recombinantly expressed via a novel cold shock expression system.The epitope profile of Ana o 3 was analyzed to identify key epitopes,and these key epitopes were fused and recombinantly expressed and used as known antigen for accurate detection of sIgE.Methods:1.Recombinant expression of rAna o 3: The total RNA extracted from cashew nut was reversely transcribed into cDNA,which was then amplified by using nested PCR.After the Ana o 3 gene was obtained,it was inserted into pCold-SUMO expression vector.The construct was identified by DNA sequencing.When expressed,the construct was transformed into BL21(DE3)competent cells,and an IPTG-induced expression was performed at 15?.After purification via a Ni-NTA column,a large quantity of rAna o 3 with good solubility was obtained.The immunocompetence of rAna o 3 was identified by western bolting,with cashew-allergic serum as a probe.2.Identification of key epitopes: Three linear epitope prediction softwares,i.e.DNAsTAR,ABCpred and Bepipred Antibody Epitope Prediction,were used to screen the key epitopes.The predicted results were comparatively analyzed with the epitopes reported from latest literature,and we decided 12 epitopes for artificial synthesis and the following verification.The IgE-binding ability of 12 epitopes was evaluated by dot blotting,using 29 Ana o 3-sIgE positive serum as primary antibody.The epitopes with high response frequency were determined as key epitopes.3.Construction,expression and purification of recombinant key epitope fusion protein: The cDNA sequences of key epitopes were linked up by flexible pepitide(GGGS),producing a multi-epitope fusion gene.This fusion gene,with Stu? and BamH? restriction enzyme sites respectively added to the 5' and 3' end,was artificially synthesized.Then,it was inserted into pCold-SUMO expression vector and the construct was verified by DNA sequencing.For expression and purification,the construct was transformed in BL21(DE3)competent cells and induced to express by an addition of IPTG.Recombinant key epitope fusion protein was purified by going through a Ni-NTA column.The immunocompetence of the protein was evaluated by western bloting.Results:1.The Ana o 3 gene was successfully cloned and inserted into pCold-SUMO expression vector.DNA sequencing results showed that the full length of Ana o 3 gene was 417 bp,encoding 138 amino acids,which was in accordance with the CDS sequence of Ana o 3 in GenBank.SDS-PAGE results indicated the molecular weight of rAna o 3 to be 27 k Da,which was in agreement with theoretical predicted value.Ana o 3 was found to have good reactivity with cashew-allergic serum.2.By bioinformatics prediction and consulting literature,12 peptides were selected as candidate epitopes.Dot bloting analysis showed that 11 of 12 epitopes had reactivity with positive sera,but of different degree.However,Peptide no.11 hardly showed any reactivity.The response frequency was calculated.It showed that Pepitide no 1,2,3,6,10 had response frequency all exceeding 80%,and thus they are counted as key epitopes.3.Considering that there were 12 amino acids overlap among peptide no.1-3,peptide no.1-3 were regarded as one epitope.The valid epitopes were linked up,recombinantly expressed and purified.SDS-PAGE results showed that the molecular weight of recombinant key epitope fusion protein was 22 kDa,which was in accordance with the theoretical predicted value.Western-bloting analysis results revealed that this fusion protein had good reactivity with Ana o 3-s IgE positive serum.Conclusion:1.A large quantity of r Ana o 3 with good immunocompetence was successfully obtained via a novel cold shock prokaryotic expression system.We have confirmed the effectiveness of the use of rAna o 3 as known antigen for cashew-s IgE detection.2.By bioinformatics prediction and consulting literature,12 candidate epitopes were obtained.Immuological analysis results showed that all epitopes except candidate no.11 were valid epitopes of Ana o 3.The prediction was relatively reliable.3.The recombinant key epitope fusion protein had good immunocompetence and could be used as high-quality antigen for determination of Ana o 3-s IgE in serum.