Study Of Crab Allergen In BALB/c Mice Model

Shellfish hypersensitivity is amongst the most common food allergies. Studies indicate that the major shellfish allergen was identified as tropomyosin (TM). Recently, arginine kinase (AK) might be a allergen in shrimp, however, whether it is a specific allergen in crab is not confirmed until now. Animal models can directly the ability of proteins to cause allergic sensitization, so we want to build mice model to assess the allergenicity of TM and AK of crab, which could supply assistance in the research of molecular mechanisms involved in crab hypersensitivity, low allergic food, and the safety and validity of allergens.Fitstly, TM of Mud crab was purified to homogeneity by isoelectric point precipitation, ammonium sulfate fractionation, and heating , and then confirmed by western-blot of Rabbit anti Chinese mitten crab TM antibody. Then we set up the mice model of TM by intraperitoneal and oral administration and compared the effect; AK of Mud crab was purified to homogeneity by the 70 % to 90 % ammonium sulfate precipitation and HiTrap Q Sepharose Fast Flow chromatography, and then confirmed by western-blot of Rabbit anti White shrimp AK antibody. Then we used two-dimensional electrophoresis to analyse its molecular weight and isoelectric point. We set up the BALB/c mice model of purified AK by intraperitoneal administration. Serum IgE was evaluated by indirect enzyme linked immunosorbent assay. Levels of IL-4, IL-13 and IFN-λwere determined from in vitro splenocyte culture upon individual allergen stimulation by sandwich-ELISA. The local and systemic anaphylactic reactions were assessed by scoring according to symptoms observed and detecting the histamine in feces. Relative expression of IL-4 mRNA was quantitatively analysed by real-time polymerase chain reaction.The molecular weight and isoelectric point of TM were 38 kDa and 4.5, respectively. The results of BALB/c mice model of TM indicated that mice from intraperitoneal and oral administration all displayed anaphylactic responese; The IgE of the former was as three times as the latter. The IL-4, IL-13 and IFN-λlevels of the former were 24.278±1.985 pg/mL, 27.776±0.0345 pg/mL, 84.243±0.0365 pg/mL, respectively. The histamine in feces was 6342.68 nM. And the latter were 17.508±2.189 pg/mL, 3.150±0.434pg/mL, 15.056±1.974 pg/mL and 6244.651 nM, respectively. In contrast, the TM-specific IgE, CD4+T cells and allergic reaction were absent in the control mice. These illustrated that both sensitized ways were suitable and the latter had a better effect. The relative expression of IL-4 mRNA of TM and OVA were 2.788 and 1.739, respectively. But there were no significant difference between them.The molecular weight and isoelectric point of purified AK of Mud crab were 40 kDa and 6.5, respectively. Compared with the negative control, the results of BALB/c mice model of AK and OVA demonstrated that all mice from intraperitoneal administration developed hypersensitive syptoms and significantly elevated IgE and histamine in feces. The spleen lymphocyte IL-4, IL-13 and IFN-λlevels were 25.859±0.072pg/mL, 22.716±0.745 pg/mL and 114.265±8.220 pg/mL. The histamine in feces was 6188.3572 nM. IL-4,IL-13 and IFN-λand histamine levels were 19.223±0.988 pg/mL, 17.292±2.831 pg/mL, 92.858±2.449 pg/mL and 6175.48 nM in OVA group, respectively. In contrast, all of them had significant difference with the. However, there was no significant difference with the positive control. The relative expression of IL-4 mRNA of AK and OVA was 1.98 and 1.739, respectively. But there were no significant difference between them.We have successfully established BALB/c mice model of TM from Mud crab by intraperitoneal and intra-gastric administration. And the former had a better effect. On this basis, we firstly built the BALB/c mice model of AK from Mud crab by intraperitoneal administration to assess its allergenicity and confirm that AK is a specific allergen of crab. This model can supply technological assisstance in the study of molecular mechanisms involved in shellfish hypersensitivity, low sensitized food , and the safety and validity of allergens.