Cao Minjie

Studies on the immunoreactivity of the major allergen tropomyosin in mantis shrimp (Squilla oratoria).

Crustacean is one of the eight kinds of allergen sources in coastal areas,which causes the IgE-mediated hypersensitive reactions with clinical manifestations including urticaria,angioedema,asthma,and even fatal anaphylaxis. Tropomyosin (TM) is the major allergen of decapod crustaceans with highly conserved amino acid sequences. The purpose of this study is to confirm whether TM is a major cross-reactive allergen in mantis shrimp (Squilla oratoria) which is taxonomically distinct from decapods and largely consumed as a delicacy in China. Quantification of TM in different species of crustaceans was also conducted. Muscle sample from mantis shrimp was homogenized with phosphate buffer to prepare heated extracts and separated by 12 % sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). A protein band with molecular mass of about 36 ku was detected by IgE-immunoblotting by all of the sera with crustacean allergy,suggesting it is the major allergen of mantis shrimp. This protein was further purified to homogeneity by acetone powder preparation,isoelectric point precipitation,ammonium sulfate fractionation (40%-60% saturation),and heat treatment. Purified protein was demonstrated to be TM by Western blot using polyclonal antibody against TM from Chinese mitten crab (Eriocheir sinensis). Heated extracts from other crustaceans including mud crab (Scylla serrata),Pacific white shrimp (Penaeus vannamei) and short necked clam (Venerupis variegata) were also prepared,and the cross-reactivity of TM from mantis shrimp with TMs from these crustaceans was confirmed by inhibition immunoblotting using polyclonal antibody against TM and inhibition ELISA using sera with crustacean allergy. Quantification by ELISA using polyclonal antibody against TM revealed that TM content in mantis shrimp muscle is much lower than that in Pacific white shrimp and mud crab muscle,which was about 45 times lower in mantis shrimp muscle than that in Pacific white shrimp muscle. However,its allergenicity seems equivalent to other decapod crustaceans as showed by inhibition ELISA using sera with crustacean allergy. This may be due to the degradation of TM in mantis shrimp by its endogenous serine proteinases and cathepsins which destroyed the IgG-binding epitope but not the IgE-binding epitopes. In conclusion,this study demonstrated that allergenicity of mantis shrimp is almost equivalent to decapods and TM is the major allergen in mantis shrimp with high cross-reactivity to decapod crustaceans.

Purification and identification of the major allergen of Mud crab.

IgE-mediated hypersensitive reactions to ingestion of crustacean are among the most serious forms of food allergies.Sensitized individuals can develop urticaria,angioedema,asthma,and even life-threatening anaphylaxis.Mud crab(Scylla serrata) is a kind of crab constitutes a promising fishery industry in China.Because of the increasing consumption of such kind of crab,the occurrence of hypersensitive reactions is extending year by year.Although tropomyosin is assumed to be a major allergen in crustaceans,few experimental data are available on allergens in crabs in China,such as Mud crab.Thus,it is important to confirm whether tropomyosin is the major allergen of Mud crab or not.In order to characterize and confirm the biochemical quality of Mud crab allergen,we report herein the isolation,identification,and determination of the major allergen of Mud crab.Crude cooked extract of Mud crab muscle was used as antigen and sera samples from 11 crustacean-allergic patients used as antibody investigated by Western-blot.Allergen with molecular mass about 38 kD was detected by all of 11 sera with crustacean allergy by Western-blot,suggesting this protein was the major allergen of Mud crab.The 38 kD protein was purified to homogeneity by isoelectric point precipitation,ammonium sulfate fractionation,and heating,and it was further characterized as tropomyosin.Polyclonal antibody against Mud crab-tropomyosin was prepared and further purified by Protein A Sepharose affinity column.The antibody cross reacted positively with tropomyosins from other crustaceans,suggesting its potential application in the detecting of the major allergen tropomyosin in crustacean foods.The identification and characterization of the major allergen in Mud crab will facilitate not only the elucidation of cross-reactions to crustaceans but also the advance of the diagnosis and treatment of seafood allergy.The 38 kD tropomyosin allergen in Mud crab as identified in the present study will benefit further allergic studies not only in this species of crab but also in other species of aquatic products.