Research On The IgE Epitope And Hypoallergenic Derivatives Of Heat-stable Allergens In Scylla Paramamosain

Mud crab（Scylla paramamosain）is an important economic aquatic product in China,which is widely popular by people because of its delicious taste and rich nutrition.However,the heat-stable allergens that still exist after thermal processing is one of the important factors causing crab allergy.Therefore,for the heat-stable allergens,it is necessary to obtain the gene sequence,characterize the physicochemical and immunological properties,map the antigen epitopes and prepare the hypoallergenic derivatives,which is of great significance to systematically understand the heat-stable allergens of crab,develop biological products for the crab allergens-specific immunotherapy,and guide the processing and production of hypoallergenic crab products.In this research,the serum pool of crab sensitised patients was used to screen the heat-stable allergens of steamed crab muscle extract,and the protein bands of 38 k Da and 18 k Da were found to have IgE binding capacity after thermal treatment.Two target proteins were purified and identified as tropomyosin（TM）and myosin light chain（MLC）by mass spectrometry.Subsequently,TM and MLC were cloned and expressed,the sequence of TM contained 852 bp,encoding 284 amino acids,and that of MLC contained 459 bp,encoding 153 amino acids.The recombinant proteins had similar secondary structure and Ig G/IgE binding capacity to the natural protein,and the recombinant protein could also significantly up-regulate the expression of CD63and CD203c on the surface of basophil in crab sensitised patients（p<0.01）.The frequency of TM and MLC bind to IgE in 177 crab sensitised patients was 32.8%and 11.9%,respectively.Based on these results,TM and MLC were named Scy p 1 and Scy p 3 by the world health organization/international union of immunological societies allergen nomenclature group,respectively.The linear mimotopes and conformational mimotopes of TM and MLC were systematically analyzed by bioinformatics software technology,phage display technology and one-bead-one-compound peptide library panning technology.Ten IgE linear epitopes of TM(A₅₉-L₇₀,A₇₆-R₉₀,R₁₀₀-T₁₁₀,L₁₁₃-R₁₂₅,M₁₂₆-E₁₃₉,R₁₄₀-Y₁₆₂,E₁₆₃-D₁₇₅,L₁₇₆-S₁₈₈,K₁₈₉-V₂₁₀,G₂₁₁-Y₂₃₁)and seven IgE linear epitopes of MLC(A₇-G₂₄,D₃₅-E₅₀,K₅₁-K₆₁,M₆₂-F₈₇,K₉₆-E₁₀₆,L₁₀₇-P₁₂₅,D₁₃₅-K₁₄₇)were obtained by synthetic peptide technology combined with inhibitory Dot blot,in which five epitopes showed stronger IgE binding capacity(TM:R₁₀₀-T₁₁₀and L₁₁₃-R₁₂₅,MLC:K₅₁-K₆₁,K₉₆-E₁₀₆and D₁₃₅-K₁₄₇).Meanwhile,the key amino acid of conformational mimotopes of TM and MLC were mutated to Ala,ten mutants（TM:K12A,E164A,Y267A,MLC:C36A,V52A,D66A,K77A,N99A,I110A,P141A）with significantly reduced IgE binding capacity were obtained,and finally verified three IgE conformational epitopes of TM and seven IgE conformational epitopes of MLC.Among them,IgE binding capacity of TM mutants（K12A and E164A）and MLC mutants（C36A and N99A）decreased more significantly,indicating that four conformational epitopes where these key amino acids are important epitopes of TM and MLC.Based on the identification of five linear epitopes with strong IgE binding capacity,two mutant derivatives（mt ALLERGEN）were obtained by the deletion of linear epitopes using genetic engineering.Four reduced and alkylationn derivatives（red/alk-wt ALLERGEN,red/alk-mt ALLERGEN）were obtained by modifying the structures of two wild-type allergens（wt ALLERGEN）and two mt ALLERGEN with iodoacetamide,respectively.IgE binding capacity and basophil activation ability of mt ALLERGEN and red/alk-mt ALLERGEN was significantly lower than that of red/alk-wt ALLERGEN,indicating that the allergenicity of wt ALLERGEN with structural damage was not obviously reduced,suggesting that the dominant IgE epitope type in TM and MLC was linear epitope.Balb/c model was used to evaluate the allergenicity of mt ALLERGEN in vivo,and it was found that compared with wt ALLERGEN,mt ALLERGEN caused mild intestinal inflammation,the number of T-helper（Th）cells and B cells in lymphocytes,and the production of specific IgE antibodies,histamine,mast cell proteases,interleukin-4,but could also significantly promote the production of interleukin-10and specific Ig G antibodies,suggesting that mt ALLERGEN equilibrated Th1 and Th2 cells in the body,and then alleviated food allergy symptoms in mice.Meanwhile,mt ALLERGEN immunized Balb/c mice and New Zealand white rabbits to produced specific Ig G antibodies can effectively block the binding of TM,MLC and patient serum IgE in vitro,indicating that the induced specific Ig G antibody had blocking ability.In conclusion,the gene sequence of heat-stable allergens were cloned in crab,recombinant proteins with similar properties to natural proteins were expressed,IgE binding frequency of heat-stable allergens were analyzed,and the world health organization/international federation of immunological societies allergen nomenclature group were approved.Ten IgE linear epitopes and three IgE conformational epitopes of TM,and seven IgE linear epitopes and seven IgE conformational epitopes of MLC were mapped by three epitopes analysis techniques,in which the epitopes with strong IgE binding capacity were identified.Using genetic engineering to delete the identification of five linear epitopes with stronger IgE binding capacity and obtain two hypoallergenic derivatives,confirming the dominant IgE epitope type in heat-stable allergens was linear epitope,elucidating the hypoallergenic derivatives brought Th1 and Th2 cells to equilibrium in vivo,and induce specific Ig G antibodies with blocking ability in vitro.These results can provide technical support for the component diagnosis of crab allergy,and lay a theoretical foundation for biological products of crab allergen-specific immunotherapy.