Antigenic Epitopes And Cross-reactivity Analysis Of Tropomyosin In Crassostrea Angulata

The prevalence of food allergies has increased in dramatic ways over the past few decades.At present,food allergy has become one of the public health problems concerned by the world.As a common shellfish product in southeast coastal areas of China,the production of oyster is increasing year by year,and the risk of food allergy caused by eating oyster is also increasing.However,there is still a lack of research on allergens in oysters.In this study,tropomyosin（TM）,an important allergen of Crassostrea angulata was purified,identified,cloned and expressed,and its physicochemical properties,antigenic epitopes and cross-reactivity were explored in order to provide theoretical basis for molecular diagnosis of oyster allergy.The allergic components of myofibrin in C.angulata were detected by the serum of oyster sensitised individuals,and a specific band with molecular weight of about 38 k Da was generated.The target protein was isolated and purified,and was identified as C.angulata TM by mass spectrometry.Native-PAGE and two-dimensional electrophoresis experiments confirmed that TM had no polymorphism and the isoelectric point was 4.6.Subsequently,TM was cloned and expressed,and the sequence contained an 852 bp open reading frame encoding 284 amino acid residues.The physicochemical properties of native TM（n TM）and recombinant TM（r TM）were further compared and analyzed.The results showed that both n TM and r TM had stable thermal stability,p H stability,digestive stability,and similar secondary structure.In addition,serological results showed that n TM and r TM had similar Ig G and Ig E-binding capacities,and could significantly up-regulate the expression levels of CD63 and CD203c molecules on the surface of basophil in oyster sensitised individuals（p<0.01）,and there was no significant difference in the ability of n TM and r TM to activate basophil.The antigenic epitopes of C.angulata TM were analyzed by bioinformatics software prediction combined with"one bead and one compound"peptide library,and verified by synthetic peptide technology combined with serological experiment.Finally,10 linear epitopes:L-TM-1(AA_15-21),L-TM-2(AA_27-41),L-TM-3(AA_43-51),L-TM-4(AA_61-76),L-TM-5(AA_84-90),L-TM-6(AA_165-174),L-TM-7(AA_182-192),L-TM-8(AA_196-207),L-TM-9(AA_215-224),L-TM-10(AA_261-268)and 2 conformational epitopes C-TM-1(K₃₆K₃₈I₃₉L₄₃L₄₆K₄₈K₄₉)and C-TM-2(K₂₆₄E₂₆₅Y₂₆₇K₂₆₈I₂₇₀E₂₇₃F₂₇₈E₂₆₃)were obtained,and the key amino acids were L₄₃ and I₂₇₀,respectively.In addition,phylogenetic tree analysis showed that TM of C.angulata was closely related with other shellfish.Meanwhile,the sequence homology in shellfish TMs were high and the epitope region was conserved.Serological test results further indicated that there were different degrees of immune cross-reactivity between C.angulata TM and other shellfish TMs.In conclusion,an important allergen TM of C.angulata was purified and identified in this study,and the recombinant protein was obtained.There were no significant differences in physicochemical and immunological properties,which improved the information of shellfish allergen.10 linear epitopes and 2 simulated conformation epitopes of portuguese oyster TM were identified,and discovered that C.angulata TM can cross-reactivity with other shellfish TM,which is expected to provide a theoretical basis for the development of hypoallergenic shellfish food and the prevention of shellfish allergy.