Study On The Allergenicity Of Sarcoplasmic Calcium-binding Protein From Scylla Paramamosain

As a kind of aquatic products with high nutritional value and tasty meat,crabs are popular with consumers.However,it is crucial to figure out that crabs,have caused numerous cases of food allergy which significantly influence human being’s health.In this study,sarcoplasmic calcium binding protein（SCP）of S.paramamosain were chosen as the research object,we mapping SCP antigen epitopes,analyze the enzymatic cross-linking reaction and maillard reaction’s effect on the allergen and further probing into the cause of the sensitization change.In this study,SCP with molecular weight of 20 kDa was purified from Scylla paramamosain.Rabbit anti-SCP-IgG was purified by specific-protein-A chromatography made from New Zealand Rabbits.The expression vector pET-22b-SCP was constructed to produce the rSCP in the Ecoli.Rossetta strain.nSCP and rSCP were present typicalα/βprotein characteristics.Both of them tend to formed dimers under the conditions of weak acid（pH 2.0⁵.0）or strong base（10.0¹1.0）.Therefore,nSCP and rSCP showed similiar immunobinding activity and structure stability characteristic.In vivo,rSCP can induce the food allergy in BALB/c mice through upregulate IL-4 and IL-13 cytokines to 300 pg/m L and 250 pg/mL respectively.In vitro,rSCP be able to increase the surface molecule CD63 level of peripheral blood eosinophilic mononuclear cells.Inhibition ELISA showed that SCP of different crustaceans or rSCP had immune cross-reaction,rSCP showed highest inhibition rate.From the above,rSCP can be instead of nSCP and used for component resolved diagnosis.Then the patients who were allergic to SCP were determined by basophil activation test,their specific IgE in serum was enrichment by anti-IgE reagent.The enriched IgE and rabbit anti-SCP-IgG were used as target protein.On the basis of three round biopanning,three IgG epitopes and six IgE epitopes were were obtained by phage display technology,which mapped to the 3D structure of S.paramamosain SCP.It was shown that overall amino acid regions of IgG epitopes overlapped with IgE epitopes amino acid regions.Antigen epitope more distribution in the conservative regional of primary sequence,α-screw terminal or flexible spatial in secondary structure and occurred in surface area of SCP.The Epitope 1(N₃₇T₃₈L₃₉I₃₈E₄₁G₄₂R₄₃Y₅₁N₅₄T₁₁₂)has highest repeatability in the cloning sequences,it may be a key region for recognization.Moreover,through the exploration of temperature,time and sugar content,we were established the maillard reaction and enzymatic cross-linking conditions（the product was named MR-SCP and CL-SCP,respectively）.After the food processing,the structure of MR-SCP and CL-SCP was changed.According to the results of circular dichroism spectrum,theα-spiral content of MR-SCP and CL-SCP were reduced to 16.4%and 16.2%,respectively,theβ-fold were increases to 32.2%and 31.3%respectively.In addition,we also tested the activation of rSCP and MR-SCP on peripheral blood eosinophils in allergic patients.It was found that rSCP could stimulate the expression of CD63⁺/CD203c⁺cell group,whereas the proportion of MR-SCP and CL-SCP stimulating activation was reduced.In-depth analysis found that the tyrosinase may catalytic crosslinking of tyrosine residues located in antigen epitope of rSCP,which influencing allergen stability and conformation and reduce its allergenic.Through the identification of the saccharification site of maillard reaction,it was found some sites were located in the epitope region of SCP,which could also form the space resistance and thus play a closed role in the antigen epitope.Based on these results,Maillard reaction or enzymatic cross-linking seemed to modify,mask or destroy SCP epitopes result to its low sensitization.