Study Of The Novel Allergens From Octopus(Octopus Fangsiao)

Seafood plays an important role in the diet of the world.The consumption of seafood products is growing rapidly because of its high nutritional value and delicious tastes.However,it may also lead to a higher frequency of reporting adverse reactions.Octopus fangsiao is a mollusc which is widely found in the ocean southeast of China.It is a popular food and an important commercial cephalopod.With the consumption of Octopus fangsiao growing rapidly,more cases of hypersensitive reaction caused by octopus have been reported.Despite the major allergen,there is little information about novel allergens in octopus.Therefore,it is significant to make research on octopus allergens.Based on previous studies,this research used octopus?Octopus fangsiao?as material to further identify novel allergens,finished its physicochemical characterization,and antigen mimotopes analysis.Furthermore processing technologies were applied to study the changes of allergen under different condition.These will provides the theoretical basis for the development of hypoallergenic octopus products,and make a great contribution to the diagnosis and treatments of octopus-allergic diseases.The mollusk-allergic patients' sera were used to confirm the 28-k Da protein's allergencity in octopus.The 28 k Da-protein was purified from octopus?Octopus fangsiao?by ammonium sulfate fractionation and a series of column chromatography?Q-Sepharose and Sephacryl S-200?.Then it was identified to be triosephosphate isomerase?TIM?by Mass Spectrometry.And then anti-octopus TIM polyclonal antibody was made by New Zealand Rabbits,the IgG was purified by protein A sepharose.The western blot analyses were performed using the serum pool of selected patients and anti-crayfish TIM polyclonal antibody to confirm its IgE and IgG binding activities.The results of its physicochemical characterization analysis showed that TIM was a glycoprotein with 1.7%?w/w?carbohydrate,the molecular weight is 28 k Da and its p I is 7.6.The full-length c DNA of TIM is 1140 bp,the complete open reading frame is 744 bp,encoding a protein composed of 247 amino acid residues and the prediction molecular weight is 26.72 k Da the prediction p I is 7.76.There is one potential N-glycosylation sites?N194A195S196?in the amino acid sequence of TIM and the active site of TIM is 162AYEPIWAIGTG172.TIM aggregated after heating above 45 °C,the aggregations still have IgG-binding activity after 60 min heating while the IgE-binding activity of TIM started to be reduced and almost disappeared at 80 °C.The purified TIM was stable in solution with different pH values,but at pH 10.0 and 11.0 the IgG-binding activity was a little fainter.High temperature treatment and extreme pH conditions?pH< 4.0,pH> 9.0?altered the secondary structure of TIM which affected the IgE-binding activity of natural TIM.TIM could be degraded into small fragments in the simulated gastric fluid digestion,but the digested fragments retained over 80% of the IgE-binding activity.TIM had the typical??/??8-barrel structure.Eight linear epitopes: L-TIM-1(N₁₄G₁₅N₁₆K₁₇K₁₈S₁₉),L-TIM-2(N₃₃A₃₄D₃₅),L-TIM-3(R₅₁S₅₂K₅₃L₅₄),L-TIM-4(S₉₅E₉₆R₉₇R₉₈),L-TIM-5(K₁₂₉L₁₃₀E₁₃₁E₁₃₂R₁₃₃E₁₃₄A₁₃₅G₁₃₆K₁₃₇T₁₃₈),L-TIM-6(H₁₅₂T₁₅₃K₁₅₄D₁₅₅W₁₅₆),L-TIM-7(N₁₉₄A₁₉₅S₁₉₆),L-TIM-8(L₂₁₉G₂₂₀Q₂₂₁K₂₂₂P₂₂₃D₂₂₄)were predicted by bioinformational method and one conformational IgG-epitope C-TIM(A₇₁G₇₄S₆₉D₇₅T₇₃F₇₂V₆₇)was identified by phage display technology.The result of sequence alignment with other alread y reported TIM as allergen shows that the homology is relatively high and the epitopes are located in the conserved domain.With the basis of octopus allergen characteristic study,maillard reaction was combined with high-temperature and high-pressure to develop a new method of hypoallergenic octopus product.The immunoreactivity of TIM,AK and TM are all been reduced.The high hydrostatic pressure was also applied to study its influence on octopus allergen.This method has a good effect on the immunoreactivity reduction of TIM and AK but failed on TM.The structure change of AK was also studied.The secondary and tertiary structure of AK was destroyed by the high hydrostatic pressure led to a result of break of epitopes.In summary,the study formed a valuable asset for its significance in allergy diagnosis of octopus-allergic disorders and supplied the technical reference for developing the hypoallergenic products of octopus.